Human cytomegalovirus (HCMV) is a ubiquitous member of the herpesvirus family that can induce a wide range of diseases, typically in newborns and immunocompromised adults. Nearly one percent of all live births in the United States are associated with congenital HCMV infection, with approximately 5 to 10 percent of infections resulting in significant neurological defects. In bone marrow transplant recipients, mortality due to HCMV pneumonia can be as high as forty percent. In addition, disseminated HCMV infection is common in immunocompromised patients, such as AIDS patients, and is frequently associated with conditions such as gastroenteritis and sight-threatening chorioretinitis.
The viral genome consists of a large double-stranded DNA molecule of approximately 230 base pairs packaged within an enveloped capsid to form the infectious virion. Productive infection is species and cell specific and requires the tightly coordinated expression of viral genes. This sequential viral gene expression is divided into three kinetic classes, immediate early (IE), early (E) and late (L). The IE gene products, which are located in four regions of the genome, are synthesized immediately after viral infection and rely primarily on host factors for their expression. The principle site of IE transcription, known as the major IE transcription unit, is in the large unique (UL) component of the genome (see, e.g., Thrower et al., J. Virol. 70:91-100, 1996; Klucher et al., Mol. Cell. Biol. 13:1238-1250, 1993; Arlt et al., J. Virol. 68:4117-4125, 1994). Early genes (such as the homolog for DNA polymerase) are transcribed prior to viral DNA replication (see, e.g., Ertl and Powell, J. Virol. 66:4126-4133, 1992; Stenberg et al., J. Virol. 63:2699-2708, 1989; He et al., J. Virol. 66:1098-1108, 1992) and the late genes, which constitute a majority of the viral genome are transcribed in abundance only after viral DNA replication (see, e.g., Depto and Stenberg, J. Virol. 66:3241-3246, 1992; Geballe et al., J. Virol. 57:864-874, 1986; Leach and Mocarski, J Virol. 63:1783-1791, 1989).
In order to successfully treat HCMV infection, sensitive and accurate diagnostic tests are required. Most current assays involve immunofluorescence techniques, which are cumbersome and often lack sensitivity. A more rapid cell-based assay is described in U.S. Pat. No. 5,418,132, but that assay is unable to distinguish among different herpesviruses. The development of diagnostic methods that detect HCMV infection rapidly and specifically would facilitate the diagnosis and treatment of HCMV infection.
In addition, conventional approaches to identifying inhibitors of viral gene expression, which typically involve the use of whole animals or tissues, are labor intensive and time consuming. Techniques involving plaque assays are more rapid, but still require on the order of two weeks for completion. A cell based viral assay has the potential for greater efficiency and sensitivity in the identification of useful therapeutic agents.
Accordingly, there is a need in the art for improved methods for diagnosing HCMV infection, and for identifying modulators of cytomegalovirus gene expression. The present invention fulfills these needs and further provides other related advantages.